Materials and methods for detecting source body fluids

ABSTRACT

The invention pertains to analyzing the levels of DNA methylation at specific genetic loci to detect specific body fluids, for example, vaginal secretions or vaginal epithelial cells, semen or sperms, saliva or buccal epithelial cells, or blood or blood cells. Particularly, the levels of methylation of DNA at the genetic loci corresponding to SEQ ID NOs: 1, 6, 11, and 16 are used to detect vaginal secretions or vaginal epithelial cells, semen or sperms, saliva or buccal epithelial cells, and blood or blood cells, respectively. The level of methylation at the specific loci can be determined by high-resolution melt analysis (HRM) or sequencing of the amplicons produced using specific primers designed to amplify the specific loci. Kits containing the primers and reagents for carrying out the methods disclosed herein are also provided.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 15/921,079, filed Mar. 14, 2018, the disclosure of which isincorporated herein by reference in its entirety, including any figures,tables, and drawings.

GOVERNMENT SUPPORT

This invention was made with government support under 2015-R2-CX-0012awarded by National Institute of Justice. The government has certainrights in the invention.

The Sequence Listing for this application is labeled“SeqList-08Mar18-ST25.txt”, which was created on Mar. 8, 2018, and is 6KB. The Sequence Listing is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

DNA is used to match a sample retrieved from a crime scene with DNAretrieved from a suspect to identify a connection of the suspect to thecrime scene. Current DNA analyses do not permit identifying the sourceof DNA from a suspect. However, certain forensic cases, such as sexualabuse require confirmation that a DNA from a suspect is from an intimatebody fluid.

Techniques currently used for body fluid identification are not based onDNA analysis. For example, microscopic observation of sperms is used toidentify semen as a source body fluid or histological staining ofglycogen-rich cells is used to identify vaginal cells. However, thesetests are not reliable. For example, if the male donor does not producesperm, the source cannot be identified as semen. Similarly, falsenegatives can occur because the glycogen content of vaginal cells variesdepending on the menstrual cycle and reproductive age; whereas, falsepositives can occur because buccal and urogenital skin cells (even frommales) can have high glycogen.

Certain other methods are based on protein/enzyme reactivity or cellstaining and are merely presumptive. These methods may have lowsensitivity and render the portion of the sample useless for subsequentanalysis. Therefore, forensic laboratories may be left to choose betweenisolating DNA to compare a suspect's DNA or determining body fluid oforigin.

Certain other methods of identifying source body fluid are based onanalyzing RNA transcripts. However, the need to identify a body fluidoften arises after DNA is isolated. To perform an RNA transcriptanalysis, the laboratory technician would have to retrieve a new portionof the original sample (if available) and isolate RNA. However, theoriginal sample may have already been consumed.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods that avoid the problems and difficultieswith current methods of detecting body fluids in a sample, particularly,a forensic sample. The methods of the invention depend on the analysesof levels of DNA methylation at specific genetic loci to detect specificbody fluids.

In one embodiment, the body fluid and/or cells present in the samplecomprise vaginal secretion or vaginal epithelial cell, semen or sperm,saliva or buccal epithelial cell, and blood or blood cell.

The level of methylation at specific loci in the genomic DNA isolatedfrom a sample can be determined by high-resolution melt analysis (HRM)of amplicons produced using specific primers designed to amplify thespecific loci.

A further embodiment of the invention provides a method for determiningthe level of methylation at specific loci in the genomic DNA isolatedfrom a cell, for example, a cell suspected to be a vaginal epithelialcell, buccal epithelial cell, sperm, or blood cell isolated from aforensic sample.

Kits containing primers and reagents for carrying out the methodsdisclosed herein are also provided.

Assays for determining the level of methylation at specific loci in thegenomic DNA isolated from a sample are also provided. In certainembodiments, the assays comprise HRM or pyrosequencing of ampliconsproduced using specific primers designed to amplify specific loci in thegenomic DNA.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 provides melt curves for the amplified region ZC3H12D, which islocated on human chromosome 6. The blue lines represent semen samplesand have a lower TM when compared to blood (red lines) and saliva (greenlines). For this marker (ZC3H12D), the typical TM for semen is 75.5°C.±0.2° C., whereas the TM for blood is 78.2° C.±0.4° C. and saliva78.1° C. with 0.3° C. SD.

FIG. 2 provides melt curves (−dF/dT) for the amplified region VE_8(cg08751438). The pink lines represent DNA samples from vaginalepithelia, blue represents semen, red represents blood and greenrepresents saliva.

FIG. 3 provides melt curves (normalized fluorescence versus temperaturein degree Celsius) for the amplified region VE_15. The pink linesrepresent DNA samples from vaginal epithelia, blue represents semen, redrepresents blood and green represents saliva.

FIG. 4 provides melt curves (normalized fluorescence versus temperaturein degree Celsius) for the amplified region INPP5D200_TM60. Orange linesrepresent DNA samples from vaginal epithelia, blue represents semen, redrepresents blood and green represents saliva.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1: Sequence of the locus specific for vaginal epithelialcell.

SEQ ID NO: 2: Sequence of a forward primer designed to amplify the locusspecific for vaginal epithelial cell.

SEQ ID NO: 3: Sequence of a reverse primer designed to amplify the locusspecific for vaginal epithelial cell.

SEQ ID NO: 4: Sequence of the locus specific for vaginal epithelial cellafter bisulfite treatment assuming 100% unmethylation of all CpG sites.

SEQ ID NO: 5: Sequence of the locus specific for vaginal epithelial cellafter bisulfite treatment assuming 100% methylation of all CpG sites.

SEQ ID NO: 6: Sequence of the locus specific for sperm.

SEQ ID NO: 7: Sequence of a forward primer designed to amplify the locusspecific for sperm.

SEQ ID NO: 8: Sequence of a reverse primer designed to amplify the locusspecific for sperm.

SEQ ID NO: 9: Sequence of the locus specific for sperm after bisulfitetreatment assuming 100% unmethylation of all CpG sites.

SEQ ID NO: 10: Sequence of the locus specific for sperm after bisulfitetreatment assuming 100% methylation of all CpG sites.

SEQ ID NO: 11: Sequence of the locus specific for blood cells.

SEQ ID NO: 12: Sequence of a forward primer designed to amplify thelocus specific for blood cells.

SEQ ID NO: 13: Sequence of a reverse primer designed to amplify thelocus specific for blood cells.

SEQ ID NO: 14: Sequence of the locus specific for blood cells afterbisulfite treatment assuming 100% unmethylation of all CpG sites.

SEQ ID NO: 15: Sequence of the locus specific for blood cells afterbisulfite treatment assuming 100% methylation of all CpG sites.

SEQ ID NO: 16: Sequence of the locus specific for saliva or buccalepithelial cells.

SEQ ID NO: 17: Sequence of a forward primer designed to amplify thelocus specific for buccal epithelial cells.

SEQ ID NO: 18: Sequence of a reverse primer designed to amplify thelocus specific for buccal epithelial cells.

SEQ ID NO: 19: Sequence of the locus specific for buccal epithelialcells after bisulfite treatment assuming 100% unmethylation of all CpGsites.

SEQ ID NO: 20: Sequence of the locus specific for buccal epithelialcells after bisulfite treatment assuming 100% methylation of all CpGsites.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides materials and methods for detecting bodyfluids in a sample comprising cells and/or body fluids, for example, aforensic sample, based on DNA analyses. DNA is commonly retrieved fromcrime scenes, and therefore, DNA isolation, storage, and general use isvalidated in criminal investigations. The methods described herein aretime and cost-effective and require little personnel training. In oneembodiment, the invention provides an assay to identify a source bodyfluid as semen, blood, saliva, or vaginal secretion in a sample.

DNA methylation is one of the epigenetic mechanisms for gene regulation.Different levels of DNA methylation in certain genetic loci control geneexpression by silencing or activating specific genes. The presence of amethyl group on the 5′ carbon of a cytosine belonging to thedinucleotide CG (CpG) is believed to prevent the binding of thetranscription machinery to the promoter of a gene. Some loci on thegenome called “tissue-specific differentially methylated regions” cantherefore be used for cell identification because they present differentlevels of DNA methylation depending on the cell studied.

In accordance with the subject invention, DNA methylation levels atspecific loci are different for certain cells from body fluids whencompared to other cells. For example, DNA methylation levels at specificloci are different for cells from blood, vaginal secretions, saliva, orsemen. Accordingly, in certain embodiments of the invention, methylationstatus at specific genetic loci in the genomic DNA is determined andused to identify the source of body fluid in a sample.

The nucleotide coordinates for genetic loci mentioned herein correspondto University of California Santa Cruz genome browser and Assembly hg19.

Identifying vaginal epithelial cells based on DNA methylation methodsprovided herein is performed by determining DNA methylation at a vaginalepithelial cell specific locus. In one embodiment, determining DNAmethylation at the vaginal epithelial cell specific locus is performedthrough the use of a specific primer pair that amplifies the vaginalepithelial cell specific locus, having the sequence of SEQ ID NO: 1,from bisulfite-treated genomic DNA. For example, an ampliconcorresponding to the vaginal epithelial cell specific locus can beobtained by PCR using bisulfite-treated genomic DNA as template and aprimer pair having the sequences of SEQ ID NOs: 2 and 3.

The vaginal epithelial cell specific locus having the sequence of SEQ IDNO: 1 is also referred to as VE_8 in this disclosure. VE_8 is a locus of131 base pairs on chromosome 16 and occupies the chromosomal locationfrom 86398381 to 86398511. As such, the genome coordinates for VE_8 arechr16: 86398381-86398511. VE_8 locus contains CpG sites that arehypomethylated in the genomes of vaginal epithelial cells compared toother cells, particularly, blood cells, semen, or buccal epithelialcells. Particularly, cytosine residues located at positions 39, 75, 87,and 97 are hypomethylated in the genomes of vaginal epithelial cellscompared to other cells, particularly, blood cells, semen, or buccalepithelial cells.

Identifying sperms based on DNA methylation methods provided herein isperformed by determining DNA methylation at a sperm specific locus. Inone embodiment, determining DNA methylation at the sperm specific locusis performed through the use of a specific primer pair that amplifies asperm specific locus, having the sequence of SEQ ID NO: 6, from thebisulfite-treated genomic DNA. An amplicon corresponding to the spermspecific locus is obtained by PCR using bisulfite-treated genomic DNA astemplate and a primer pair having the sequences of SEQ ID NOs: 7 and 8.

The sperm specific locus having the sequence of SEQ ID NO: 6 is alsoreferred to as ZC3H12D in this disclosure. ZC3H12D is a locus of 91 basepairs on chromosome 6 and occupies the chromosomal location from149778061 to 149778151. The genome coordinates for ZC3H12D arechr6:149778061-149778151. ZC3H12D locus contains CpG sites that arehypomethylated in the genomes of sperms compared to other cells,particularly, blood cells, vaginal epithelial cells, or buccalepithelial cells. Particularly, cytosine residues located at positions21, 43, 45, 55, 62, and 67 are hypomethylated in the genomes of vaginalepithelial cells compared to other cells, particularly, blood cells,semen, or buccal epithelial cells.

Identifying blood cells based on DNA methylation methods provided hereinis performed by determining DNA methylation at a blood cell specificlocus. In one embodiment, determining DNA methylation at the blood cellspecific locus is performed through the use of a specific primer pairthat amplifies a blood cell specific locus, having the sequence of SEQID NO: 11, from the bisulfite-treated genomic DNA. An ampliconcorresponding to the blood cell specific locus is obtained by PCR usingbisulfite-treated genomic DNA as template and a primer pair having thesequences of SEQ ID NOs: 12 and 13.

The blood cell specific locus having the sequence of SEQ ID NO: 11 isalso referred to as INPP5D200_TM60 in this disclosure. INPP5D200_TM60 isa locus of 156 base pairs on chromosome 2 and occupies the chromosomallocation from 233060162 to 233060317. The genome coordinates forINPP5D200_TM60 are chr2: 233060162-233060317. INPD200_TM60 locuscontains CpG sites that are hypomethylated in the genomes of bloodcells, particularly, white blood cells, compared to other cells,particularly, vaginal epithelial cells, semen, or buccal epithelialcells. Particularly, cytosine residues located at positions 24, 26, 33,59, and 130 are hypomethylated in the genomes of vaginal epithelialcells compared to other cells, particularly, blood cells, semen, orbuccal epithelial cells.

Identifying buccal epithelial cells based on DNA methylation methodsprovided herein is performed by determining DNA methylation at a buccalepithelial cell specific locus. In one embodiment, determining DNAmethylation at the buccal epithelial cell specific locus is performedthrough the use of a specific primer pair that amplifies a buccalepithelial cell specific locus, having the sequence of SEQ ID NO: 16,from the bisulfite-treated genomic DNA. An amplicon corresponding to thebuccal epithelial cell specific locus is obtained by PCR usingbisulfite-treated genomic DNA as template and a primer pair having thesequences of SEQ ID NOs: 17 and 18.

The buccal epithelial cell specific locus having the sequence of SEQ IDNO: 16 is also referred to as VE_15 in this disclosure. VE_15 is a locusof 199 base pairs on chromosome 7 and occupies the chromosomal locationfrom 27199577 to 27199775. The genome coordinates for VE_15 arechr6:27199577-27199775. VE_15 locus contains CpG sites that arehypomethylated in the genomes of sperms compared to other cells,particularly, blood cells, vaginal epithelial cells, or buccalepithelial cells. Particularly, cytosine residues located at positions27, 68, 86, 91, 125, 150, 166 and 176 are hypomethylated in the genomesof vaginal epithelial cells compared to other cells, particularly, bloodcells, semen, or buccal epithelial cells.

The methods described herein can be practiced with minute amounts ofgenomic DNA, for example, between 1 ng to 50 ng, particularly, between 5ng to 30 ng, more particularly, at about 20 ng. Moreover, methylationlevels at specific loci described herein, for example, SEQ ID NOs: 1, 6,11, and 16, can be performed when mixtures of body fluids are present.

Accordingly, one embodiment of the invention provides a method foridentifying a sample as containing, or not containing, one or more cellsselected from a vaginal epithelial cell, a sperm, a blood cell, or abuccal epithelial cell, the method comprising the steps of:

-   -   a) determining the level of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, 11, and 16,        in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the levels of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, 11, and 16;        and    -   c) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 1 in the genomic DNA isolated            from the sample,        -   ii) containing or not containing the sperm based on the            level of methylation at the genetic locus corresponding to            SEQ ID NO: 6 in the genomic DNA isolated from the sample,        -   iii) containing or not containing the blood cell based on            the level of methylation at the genetic locus corresponding            to SEQ ID NO: 11 in the genomic DNA isolated from the            sample, and/or        -   iv) containing or not containing the buccal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 16 in the genomic DNA isolated            from the sample.

In certain embodiments, the methods described herein to identify asample as containing a vaginal epithelial cell, sperm, blood cell, or abuccal epithelial cell are practiced on a forensic sample to detect thepresence of one or more of these cells in the forensic sample. Incertain embodiments, the methods are practiced on a forensic sample thatis processed to separate a cell suspected to be a vaginal epithelialcell, sperm, blood cell, or buccal epithelial cell before the step ofisolating the genomic DNA. In an even further embodiment, the methodsare practiced on a forensic sample that is known to contain only vaginalepithelial cells, sperms, blood cells, or buccal epithelial cells or acombination thereof.

In one embodiment, the invention provides a method for identifying asample as containing, or not containing, a vaginal epithelial cell, themethod comprising the steps of:

-   -   a) determining the level of methylation at the genetic locus        corresponding to SEQ ID NO: 1 in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the level of methylation at the genetic locus        corresponding to SEQ ID NO: 1; and    -   c) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 1 in the genomic DNA isolated            from the sample.

In certain such embodiments, the methods further comprise identifying asample as containing, or not containing, one or more cells selected froma sperm, a blood cell, and a buccal epithelial cell, the methodcomprising the steps of:

-   -   a) determining the level of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 6, 11, and 16, in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the levels of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 6, 11, and 16; and    -   c) identifying the sample as:        -   i) containing or not containing the sperm based on the level            of methylation at the genetic locus corresponding to SEQ ID            NO: 6 in the genomic DNA isolated from the sample,        -   ii) containing or not containing the blood cell based on the            level of methylation at the genetic locus corresponding to            SEQ ID NO: 11 in the genomic DNA isolated from the sample,            and/or        -   iii) containing or not containing the buccal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 16 in the genomic DNA isolated            from the sample.

In another embodiment, the invention provides a method for identifying asample as containing, or not containing, a sperm, the method comprisingthe steps of:

-   -   a) determining the level of methylation at the genetic locus        corresponding to SEQ ID NO: 6 in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the level of methylation at the genetic locus        corresponding to SEQ ID NO: 6; and    -   c) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 6 in the genomic DNA isolated            from the sample.

In certain such embodiments, the methods further comprise identifying asample as containing, or not containing, one or more cells selected froma vaginal epithelial cell, a blood cell, and a buccal epithelial cell,the method comprising the steps of:

-   -   a) determining the level of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 11, and 16, in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the levels of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 11, and 16; and    -   c) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 1 in the genomic DNA isolated            from the sample,        -   ii) containing or not containing the blood cell based on the            level of methylation at the genetic locus corresponding to            SEQ ID NO: 11 in the genomic DNA isolated from the sample,            and/or        -   iii) containing or not containing the buccal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 16 in the genomic DNA isolated            from the sample.

In a further embodiment, the invention provides a method for identifyinga sample as containing, or not containing, a blood cell, the methodcomprising the steps of:

-   -   a) determining the level of methylation at the genetic locus        corresponding to SEQ ID NO: 11 in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the level of methylation at the genetic locus        corresponding to SEQ ID NO: 11; and    -   c) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 11 in the genomic DNA isolated            from the sample.

In certain such embodiments, the methods further comprise identifying asample as containing, or not containing, one or more cells selected froma vaginal epithelial cell, a sperm, and a buccal epithelial cell, themethod comprising the steps of:

-   -   a) determining the level of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, and 16, in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the levels of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, and 16; and    -   c) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 1 in the genomic DNA isolated            from the sample,        -   ii) containing or not containing the sperm based on the            level of methylation at the genetic locus corresponding to            SEQ ID NO: 6 in the genomic DNA isolated from the sample,            and/or        -   iii) containing or not containing the buccal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 16 in the genomic DNA isolated            from the sample.

In an even further embodiment, the invention provides a method foridentifying a sample as containing, or not containing, a buccalepithelial cell, the method comprising the steps of:

-   -   a) determining the level of methylation at the genetic locus        corresponding to SEQ ID NO: 16 in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the level of methylation at the genetic locus        corresponding to SEQ ID NO: 16; and    -   c) identifying the sample as:        -   i) containing or not containing the buccal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 16 in the genomic DNA isolated            from the sample.

In certain such embodiments, the methods further comprise identifying asample as containing, or not containing, one or more cells selected froma vaginal epithelial cell, a sperm, and a blood cell, the methodcomprising the steps of:

-   -   a) determining the level of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, and 11, in:        -   i) a genomic DNA isolated from the sample, and        -   ii) optionally, a control genomic DNA;    -   b) optionally, obtaining one or more reference values        corresponding to the levels of methylation at the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, and 11; and    -   c) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the level of methylation at the genetic locus            corresponding to SEQ ID NO: 1 in the genomic DNA isolated            from the sample,        -   ii) containing or not containing the sperm based on the            level of methylation at the genetic locus corresponding to            SEQ ID NO: 6 in the genomic DNA isolated from the sample,            and/or        -   iii) containing or not containing the blood cell based on            the level of methylation at the genetic locus corresponding            to SEQ ID NO: 11 in the genomic DNA isolated from the            sample.            In certain embodiments, the methods described herein to            identify a sample as containing a vaginal epithelial cell,            sperm, blood cell, a buccal epithelial cell, or a            combination thereof are practiced on a forensic sample to            detect the presence of one or more of these cells in the            forensic sample. In certain embodiments, the methods are            practiced on a forensic sample that is processed to separate            a cell suspected to be a vaginal epithelial cell, sperm,            blood cell, buccal epithelial cell, or a combination thereof            before the step of isolating the genomic DNA. In an even            further embodiment, the methods are practiced on a forensic            sample that is known to contain only vaginal epithelial            cells, sperms, blood cells, buccal epithelial cells, or a            combination thereof. The control sample used in the methods            of the invention can be obtained from one or more of the            following: a known vaginal epithelial cell, a known sperm, a            known blood cell, and a known buccal epithelial cell. The            control sample can also be a cell other than a vaginal            epithelial cell, sperm, blood cell, or buccal epithelial            cell and that is known to have methylation levels at the            specific genetic loci corresponding to SEQ ID NOs: 1, 6, 11,            and 16 to be different from the methylation levels at SEQ ID            NOs: 1, 6, 11, and 16 from a vaginal epithelial cell, sperm,            blood cell, and buccal epithelial cell, respectively.

If the control sample is a vaginal epithelial cell, the step ofidentifying the sample as containing the vaginal epithelial cell isbased on the level of methylation at the genetic locus corresponding toSEQ ID NO: 1 in the genomic DNA isolated from the sample being similarto the level of methylation at the genetic locus corresponding to SEQ IDNO: 1 in the control genomic DNA. Also, if the control sample is avaginal epithelial cell, the step of identifying the sample as notcontaining the vaginal epithelial cell is based on the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 1 in thegenomic DNA isolated from the sample being different from the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 1 in thecontrol genomic DNA.

If the control sample is a cell different from a vaginal epithelialcell, the step of identifying the sample as containing the vaginalepithelial cell is based on the level of methylation at the geneticlocus corresponding to SEQ ID NO: 1 in the genomic DNA isolated from thesample being different from the level of methylation at the geneticlocus corresponding to SEQ ID NO: 1 in the control genomic DNA. Also, ifthe control sample is a cell different from a vaginal epithelial cell,the step of identifying the sample as not containing the vaginalepithelial cell is based on the level of methylation at the geneticlocus corresponding to SEQ ID NO: 1 in the genomic DNA isolated from thesample being similar to the level of methylation at the genetic locuscorresponding to SEQ ID NO: 1 in the control genomic DNA.

The reference value corresponding to the level of methylation at thegenetic locus corresponding to SEQ ID NO: 1 can indicate the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 1 in avaginal epithelial cell or a cell other than a vaginal epithelial cell.As such, the reference value corresponding to level of methylation atthe genetic locus corresponding to SEQ ID NO: 1 can indicate thepresence or absence of a vaginal epithelial cell. In a vaginalepithelial cell, the genetic locus VE_8 is methylated at less than about20%, less than about 15%, less than about 10%, less than about 5%, orless than about 1%.

Similarly, if the control sample is a sperm, the step of identifying thesample as containing the sperm is based on the level of methylation atthe genetic locus corresponding to SEQ ID NO: 6 in the genomic DNAisolated from the sample being similar to the level of methylation atthe genetic locus corresponding to SEQ ID NO: 6 in the control genomicDNA. Also, if the control sample is a sperm, the step of identifying thesample as not containing the sperm is based on the level of methylationat the genetic locus corresponding to SEQ ID NO: 6 in the genomic DNAisolated from the sample being different from the level of methylationat the genetic locus corresponding to SEQ ID NO: 6 in the controlgenomic DNA.

If the control sample is a cell different from a sperm, the step ofidentifying the sample as containing the sperm is based on the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 6 in thegenomic DNA isolated from the sample being different from the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 6 in thecontrol genomic DNA. Also, if the control sample is a cell differentfrom a sperm, the step of identifying the sample as not containing thesperm is based on the level of methylation at the genetic locuscorresponding to SEQ ID NO: 6 in the genomic DNA isolated from thesample being similar to the level of methylation at the genetic locuscorresponding to SEQ ID NO: 6 in the control genomic DNA.

The reference value corresponding to the level of methylation at thegenetic locus corresponding to SEQ ID NO: 6 can indicate the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 6 in asperm or a cell other than a sperm. As such, the reference valuecorresponding to level of methylation at the genetic locus correspondingto SEQ ID NO: 6 can indicate the presence or absence of a sperm. In asperm, the genetic locus ZC3H12D is methylated at less than about 20%,less than about 15%, less than about 10%, less than about 5%, or lessthan about 1%

Further, if the control sample is a blood cell, the step of identifyingthe sample as containing the blood cell is based on the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 11 in thegenomic DNA isolated from the sample being similar to the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 11 in thecontrol genomic DNA. Also, if the control sample is a blood cell, thestep of identifying the sample as not containing the blood cell is basedon the level of methylation at the genetic locus corresponding to SEQ IDNO: 11 in the genomic DNA isolated from the sample being different fromthe level of methylation at the genetic locus corresponding to SEQ IDNO: 11 in the control genomic DNA.

If the control sample is a cell different from a blood cell, the step ofidentifying the sample as containing the blood cell is based on thelevel of methylation at the genetic locus corresponding to SEQ ID NO: 11in the genomic DNA isolated from the sample being different from thelevel of methylation at the genetic locus corresponding to SEQ ID NO: 11in the control genomic DNA. Also, if the control sample is a celldifferent from a blood cell, the step of identifying the sample as notcontaining the blood cell is based on the level of methylation at thegenetic locus corresponding to SEQ ID NO: 11 in the genomic DNA isolatedfrom the sample being similar to the level of methylation at the geneticlocus corresponding to SEQ ID NO: 11 in the control genomic DNA.

The reference value corresponding to the level of methylation at thegenetic locus corresponding to SEQ ID NO: 11 can indicate the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 11 in ablood cell or a cell other than a blood cell. As such, the referencevalue corresponding to level of methylation at the genetic locuscorresponding to SEQ ID NO: 11 can indicate the presence or absence of ablood cell. In a blood cell, the genetic locus INPD200_TM60 ismethylated at less than about 20%, less than about 15%, less than about10%, less than about 5%, or less than about 1%.

Furthermore, if the control sample is a buccal epithelial cell, the stepof identifying the sample as containing the buccal epithelial cell isbased on the level of methylation at the genetic locus corresponding toSEQ ID NO: 16 in the genomic DNA isolated from the sample being similarto the level of methylation at the genetic locus corresponding to SEQ IDNO: 16 in the control genomic DNA. Also, if the control sample is abuccal epithelial cell, the step of identifying the sample as notcontaining the buccal epithelial cell is based on the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 16 in thegenomic DNA isolated from the sample being different from the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 16 in thecontrol genomic DNA.

If the control sample is a cell different from a buccal epithelial cell,the step of identifying the sample as containing the buccal epithelialcell is based on the level of methylation at the genetic locuscorresponding to SEQ ID NO: 16 in the genomic DNA isolated from thesample being different from the level of methylation at the geneticlocus corresponding to SEQ ID NO: 16 in the control genomic DNA. Also,if the control sample is a cell different from a buccal epithelial cell,the step of identifying the sample as not containing the buccalepithelial cell is based on the level of methylation at the geneticlocus corresponding to SEQ ID NO: 16 in the genomic DNA isolated fromthe sample being similar to the level of methylation at the geneticlocus corresponding to SEQ ID NO: 16 in the control genomic DNA.

The reference value corresponding to the level of methylation at thegenetic locus corresponding to SEQ ID NO: 16 can indicate the level ofmethylation at the genetic locus corresponding to SEQ ID NO: 16 in abuccal epithelial cell or a cell other than a buccal epithelial cell. Assuch, the reference value corresponding to level of methylation at thegenetic locus corresponding to SEQ ID NO: 16 can indicate the presenceor absence of a buccal epithelial cell. In a buccal epithelial cell, thegenetic locus VE_15 is methylated at less than about 20%, less thanabout 15%, less than about 10%, less than about 5%, or less than about1%.

In one embodiment, the level of methylation of specific genetic loci areused to determine the purity of a preparation of a cell comprising orconsisting of one or more of a vaginal epithelial cell, sperm, bloodcell, and buccal epithelial cell.

For example, if a vaginal epithelial cell sample is obtained, the levelof methylation at the VE_8 locus can be checked in the sample toidentify the purity of the vaginal epithelial cells in the sample. Forexample, if the VE_8 genetic locus is about 100% un-methylated, thevaginal epithelial cell sample is almost 100% pure; whereas, if the VE_8genetic locus is only about 50% un-methylated, the vaginal epithelialcell sample is not pure and may contain 50% of other types cells,particularly, sperms, blood cells, or buccal epithelial cells.

Similarly, if a sperm sample is obtained, the level of methylation atthe ZC3H12D locus can be checked in the sample to identify the purity ofthe sperm sample. For example, if the ZC3H12D genetic locus is about100% un-methylated, the sperm sample is almost 100% pure; whereas, ifthe ZC3H12D genetic locus is only about 50% un-methylated, the spermsample is not pure and may contain 50% of other types cells,particularly, vaginal epithelial cells, blood cells, or buccalepithelial cells.

Further, if a blood cell sample is obtained, the level of methylation atthe INPP5D200_TM60 locus can be checked in the sample to identify thepurity of the blood cell sample. For example, if the INPP5D200_TM60genetic locus is about 100% un-methylated, the blood cell sample isalmost 100% pure; whereas, if the INPP5D200_TM60 genetic locus is onlyabout 50% un-methylated, the blood cell sample is not pure and maycontain 50% of other types cells, particularly, vaginal epithelialcells, sperms, or buccal epithelial cells.

Furthermore, if a buccal epithelial cell sample is obtained, the levelof methylation at the VE_15 locus can be checked in the sample toidentify the purity of the buccal epithelial cell sample. For example,if the VE_15 genetic locus is about 100% un-methylated, the buccalepithelial cell sample is almost 100% pure; whereas, if the VE_15genetic locus is only about 50% un-methylated, the buccal epithelialcell sample is not pure and may contain 50% of other types cells,particularly, vaginal epithelial cells, sperms, or blood cells.

Various techniques are known to a person of ordinary skill in the art todetermine the level of methylation at the specific loci in a genomicDNA. Non-limiting examples of such techniques include bisulfiteconversion, HRM, digestion by restriction enzymes followed by PCR,Combined Bisulfite Restriction Analysis (COBRA), direct sequencing,cloning and sequencing, bisulfite treatment and sequences, bisulfitetreatment and pyrosequencing, mass spectrometry analysis orprobe/microarray based assay. Certain techniques of determiningmethylation at certain genomic sites are described in Eads et al., Xionget al., Paul et al., Warnecke et al., Tost et al., and Ehrich et al.,the contents of which are herein incorporated in their entirety.Additional techniques for determining the level of methylation at agenetic are known to a person of ordinary skill in the art and suchtechniques are within the purview of the invention.

In a particular embodiment, the levels of methylation at the specificloci in a genomic DNA obtained from a sample are determined by HRM. HRMinvolves bisulfite treatment of genomic DNA. Bisulfite treatment ofgenomic DNA chemically changes the unmethylated cytosines to uracil,while the methylated cytosines are unchanged. In the PCR reaction usingprimers designed to amplify a specific locus, uracils are copied asthymines making the PCR product produced from an un-methylated locusthymine-rich, thereby reducing the melting temperature (TM) of theamplicon compared to the PCR product produced from a methylated locus,which is cytosine-rich. As such, methylation of the specific locus canbe determined based on TM of the amplicon, i.e., higher TM indicates amethylated locus and lower TM indicates an un-methylated locus. Thistechnique is referred HRM.

Determining the levels of methylation at the specific loci in a genomicDNA obtained from a sample by HRM comprises the steps of: obtaining thesample, isolating genomic DNA from the sample, treating the isolated DNAwith bisulfite, PCR amplifying the genetic loci using specificallydesigned primers to produce amplicons corresponding to the genetic loci,determining the melting temperatures of the amplicons produced in thePCR, determining the levels of methylation at the specific loci in agenomic DNA based on the melting temperatures of the amplicons. Themethylation status at the specific genetic loci can be used to identifythe source of body fluid based.

In one embodiment of the invention, amplification and melt analysis areperformed in a single instrument, namely, a real time PCR instrumentwith melt capacity. Use of a single instrument diminishes the hands-ontime making the methods efficient.

In one embodiment, a primer pair designed to amplify the genetic locuscorresponding to SEQ ID NO: 1 comprises a forward primer comprising SEQID NO: 2 and a reverse primer comprising SEQ ID NO: 3. A skilled artisancan design primer pairs other than SEQ ID NOs: 2 and 3 to amplify thegenetic locus VE_8 based on the sequence of SEQ ID NO: 1 and thesequences of the regions flanking VE_8 in the genomic DNA. Suchembodiments are within the purview of the invention.

In another embodiment, a primer pair designed to amplify the geneticlocus corresponding to SEQ ID NO: 6 comprises a forward primercomprising SEQ ID NO: 7 and a reverse primer comprising SEQ ID NO: 8. Askilled artisan can design primer pairs other than SEQ ID NOs: 7 and 8to amplify the genetic locus ZC3H12D based on the sequence of SEQ ID NO:6 and the sequences of the regions flanking ZC3H12D in the genomic DNA.Such embodiments are within the purview of the invention.

In a further embodiment, a primer pair designed to amplify the geneticlocus corresponding to SEQ ID NOs: 11 comprises a forward primercomprising SEQ ID NO: 12 and a reverse primer comprising SEQ ID NO: 13.A skilled artisan can design primer pairs other than SEQ ID NOs: 12 and13 to amplify the genetic locus INPD200_TM60 based on the sequences ofSEQ ID NOs: 11 and the sequences of regions flanking INPD200_TM60 in thegenomic DNA. Such embodiments are within the purview of the invention.

In an even further embodiment, a primer pair designed to amplify thegenetic locus corresponding to SEQ ID NOs: 16 comprises a forward primercomprising SEQ ID NO: 17 and a reverse primer comprising SEQ ID NO: 18.A skilled artisan can design primer pairs other than SEQ ID NOs: 17 and18 to amplify the genetic locus VE_15 based on the sequence of SEQ IDNOs: 16 and the sequences of the regions flanking VE_15 in the genomicDNA. Such embodiments are within the purview of the invention.

Accordingly, in one embodiment, the invention provides a method foridentifying a sample as containing, or not containing, one or more cellsselected from a vaginal epithelial cell, a sperm, a blood cell, or abuccal epithelial cell, the method comprising the steps of:

-   -   a) isolating genomic DNA from the sample and optionally, a        control sample;    -   b) treating the isolated genomic DNA with bisulfite;    -   c) PCR amplifying the genetic loci corresponding to one or more        of SEQ ID NOs: 1, 6, 11, and 16 to produce the corresponding one        or more amplicons, wherein the PCR amplifying is performed using        one or more primer pairs selected from:        -   i) SEQ ID NOs: 2 and 3,        -   ii) SEQ ID NOs: 6 and 7,        -   iii) SEQ ID NOs: 12 and 13, or        -   iv) SEQ ID NOs: 17 and 18; and    -   d) determining the melting temperatures of the one or more        amplicons; and    -   e) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the melting temperature of the amplicon            corresponding to the genetic locus corresponding to SEQ ID            NO: 1 in the genomic DNA isolated from the sample,        -   ii) containing or not containing the sperm based on the            melting temperature of the amplicon corresponding to the            genetic locus corresponding to SEQ ID NO: 6 in the genomic            DNA isolated from the sample,        -   iii) containing or not containing the blood cell based on            the melting temperature of the amplicon corresponding to the            genetic locus corresponding to SEQ ID NO: 11 in the genomic            DNA isolated from the sample, and/or        -   iv) containing or not containing the buccal epithelial cell            based on the melting temperature of the amplicon            corresponding to the genetic locus corresponding to SEQ ID            NO: 16 in the genomic DNA isolated from the sample.

In a specific embodiment, the invention provides a method foridentifying a sample as containing, or not containing a vaginalepithelial cell based on the methylation level of SEQ ID NO: 1determined using the primer pair of SEQ ID NO: 2 and 3. Such methods canfurther comprise identifying a sample as containing, or not containing,one or more cells selected from a sperm, a blood cell, and a buccalepithelial cell, based on the methylation level of one or more of SEQ IDNOs: 6, 11, and 16 determined using the primer pairs of SEQ ID NOs: 6and 7, 12 and 13, and 17 and 18, respectively.

In another embodiment, the invention provides a method for identifying asample as containing, or not containing a sperm based on the methylationlevel of SEQ ID NO: 6 determined using the primer pair of SEQ ID NO: 6and 7. Such methods can further comprise identifying a sample ascontaining, or not containing, one or more cells selected from a vaginalepithelial cell, a blood cell, and a buccal epithelial cell, based onthe methylation level of one or more of SEQ ID NOs: 1, 11, and 16determined using the primer pairs of SEQ ID NOs: 2 and 3, 12 and 13, and17 and 18, respectively.

In a further embodiment, the invention provides a method for identifyinga sample as containing, or not containing a blood cell based on themethylation level of SEQ ID NO: 11 determined using the primer pair ofSEQ ID NO: 12 and 13. Such methods can further comprise identifying asample as containing, or not containing, one or more cells selected froma vaginal epithelial cell, a sperm, and a buccal epithelial cell, basedon the methylation level of one or more of SEQ ID NOs: 1, 6, and 16determined using the primer pairs of SEQ ID NOs: 2 and 3, 6 and 7, and17 and 18, respectively.

In an even further embodiment, the invention provides a method foridentifying a sample as containing, or not containing a buccalepithelial cell based on the methylation level of SEQ ID NO: 16determined using the primer pair of SEQ ID NO: 17 and 18. Such methodscan further comprise identifying a sample as containing, or notcontaining, one or more cells selected from a vaginal epithelial cell, asperm, and a blood cell based on the methylation level of one or more ofSEQ ID NOs: 1, 6, and 11 determined using the primer pairs of SEQ IDNOs: 2 and 3, 6 and 7, and 12 and 13, respectively.

In certain embodiments, the methods described herein to identify asample as containing a vaginal epithelial cell, sperm, blood cell, or abuccal epithelial cell are practiced on a forensic sample to detect thepresence of one or more of these cells in the forensic sample. Incertain embodiments, the methods are practiced on a forensic sample thatis processed to separate a cell suspected to be a vaginal epithelialcell, sperm, blood cell, or buccal epithelial cell before the step ofisolating the genomic DNA.

In one embodiment, the melting temperature of one or more ampliconscorresponding to the genetic loci corresponding to SEQ ID NOs: 1, 6, 11,and 16 are compared to a reference value to determine the methylationstatus of the genetic loci corresponding to SEQ ID NOs: 1, 6, 11, and16, which in turn is used to identify the sample as containing or notcontaining one or more cells selected from a vaginal epithelial cell, asperm, a blood cell, or a buccal epithelial cell.

In one embodiment, the melting temperature of about 76.6° C. for theamplicon corresponding to the genetic loci corresponding to SEQ ID NO: 1is used to identify the presence of a vaginal epithelial cell in thesample; whereas, the melting temperature of about 78.1° C. for theamplicon corresponding to the genetic loci corresponding to SEQ ID NO: 1is used to identify the absence of a vaginal epithelial cell in thesample.

In another embodiment, the melting temperature of about 75.5° C. for theamplicon corresponding to the genetic loci corresponding to SEQ ID NO: 6is used to identify the presence of a sperm in the sample; whereas, themelting temperature of about 78.2° C. for the amplicon corresponding tothe genetic loci corresponding to SEQ ID NO: 1 is used to identify theabsence of a sperm in the sample.

In a further embodiment, the melting temperature of about 80° C. for theamplicon corresponding to the genetic loci corresponding to SEQ ID NO:11 is used to identify the presence of a blood cell in the sample;whereas, the melting temperature of about 83° C. for the ampliconcorresponding to the genetic loci corresponding to SEQ ID NO: 11 is usedto identify the absence of a blood cell in the sample.

In an even further embodiment, the identification of saliva is madedepending on the shape of the normalized curve for the graph offluorescence versus temperature in a melting curve analysis of theamplicon corresponding to the genetic loci corresponding to SEQ ID NO:16. Typically, unmethylated and methylated controls are run in a meltingcurve analysis of a sample and a saliva sample displays a curve that isintermediate between the unmethylated control and methylated control.The other body fluids and cells, for example, semen, blood, and vaginalepithelial cells show a curve similar to unmethylated control (FIG. 3).

The four primer sets described above would amplify genomic DNA isolatedfrom samples containing one or more of vaginal epithelial cells orvaginal secretions, sperms or semen, blood or blood cells, and buccalepithelial cell or saliva. The identification of different body fluidsis made only after amplification and further analysis, for example, HRMor sequencing analysis.

The primer pairs amplify bisulfite treated genomic DNA regardless of itsmethylation status and would not amplify genomic DNA that is notbisulfite treated. Therefore, if the DNA sample is not appropriate forPCR amplification, for example, due to a low amount of DNA or presenceof impurities, the amplicons will not be produced.

A control DNA sample added can be used and treated in the same manner asa test sample to ensure that the reagents are working properly.Therefore, if a test sample fails to produce amplicons, the sample canbe identified as a source of a problem for lack of amplification despitehaving working reagents. Likewise a negative control can be run wherewater replaces the genomic DNA to ensure that any amplification is notdue to unspecific amplification arising from contaminated reagents.

A control primer pair can also be used that would amplify genomic DNAthat is not bisulfite converted. The presence of an amplicon for thisprimer pair would tell a user that the bisulfite conversion was notsuccessful. Therefore, instead of having an amplification that did notwork for an unknown reason, a user would identify a failed bisulfiteconversion.

In one embodiment, an amplicon corresponding to a genetic locus isdistinguished from other amplicons based on a fluorophore attached tothe primer pair designed to amplify the genetic locus. Differentfluorophores attached to different primer pairs can be detected atdifferent wavelengths using fluorescence detectors.

In one embodiment, each amplicon is labelled with a fluorophore anddifferent fluorophores are matched with different melt curves. Bymatching fluorophores with melt curves, the presence of different bodyfluids in a sample containing a mixture of body fluids can be detected.

In another embodiment, each primer pair is labelled with a fluorophorethat is detected at a specific wavelength. As the multiplex PCRproceeds, the fluorophore in the primers is quenched by a nucleotidethat specifically binds to the DNA amplicon. Fluorescence decreases withthe increase of amplification for that specific locus.

An example of detecting different fluorophores for different primerpairs is provided in the Plexor® qPCR and RT-PCR from Promega, Inc.Certain details of Plexor® qPCR are provided in Technical Manual forPlexor® qPCR (Promega, Literature # TM262, Revised 09/09), the contentsof which are herein incorporated by reference in its entirety.

In certain embodiments, a control genomic DNA can comprise DNA that isnot bisulfite treated. Therefore, each locus has a “control primer set”directed to a bisulfite untreated genomic DNA sequence and a “testprimer sequence” directed to bisulfite treated genomic DNA. Therefore,for every test, one can have a multiplex primer set directed to abisulfite treated test genomic DNA and a multiplex primer set directedto a bisulfite untreated control genomic DNA.

In a particular embodiment, the levels of methylation at the specificloci in a genomic DNA obtained from a sample are performed bysequencing, for example, pyrosequencing. Determining methylation of agenetic locus based on sequencing involves bisulfite treating a genomicDNA. Bisulfate treatment of genomic DNA chemically changes theunmethylated cytosines to uracil, while the methylated cytosines areunchanged. In the PCR reaction using primers designed to amplify aspecific locus, uracils are copied as thymines in an un-methylated locusand cytosines are copied as guanines. As such, methylation of thespecific locus can be determined based on the presence of cytosine inthe amplicon; whereas, lack of methylation can be determined based onthe presence of cytosine in the amplicon.

Determining the levels of methylation at the specific loci in a genomicDNA obtained from a sample by sequencing comprises the steps of:obtaining the sample, isolating genomic DNA from the sample, treatingthe isolated DNA with bisulfite, PCR amplifying the genetic loci usingspecifically designed primers to produce amplicons corresponding to thegenetic loci, determining the sequences of the amplicons produced in thePCR, and determining the levels of methylation at the specific loci in agenomic DNA based on sequences of the amplicons. The methylation statusat the specific genetic loci can be used to identify the source of bodyfluid based.

Accordingly, in one embodiment, the invention provides a method foridentifying a sample as containing or not containing one or more cellsselected from a vaginal epithelial cell, a sperm, a blood cell, or abuccal epithelial cell, the method comprising the steps of:

-   -   a) isolating genomic DNA from the sample and optionally, a        control sample;    -   b) treating the isolated genomic DNA with bisulfite;    -   c) PCR amplifying the genetic loci genetic loci corresponding to        one or more of SEQ ID NOs: 1, 6, 11, and 16 to produce the        corresponding one or more amplicons, wherein the PCR amplifying        is performed using one or more primer pairs selected from        -   i) SEQ ID NOs: 2 and 3,        -   ii) SEQ ID NOs: 6 and 7,        -   iii) SEQ ID NOs: 12 and 13, or        -   iv) SEQ ID NOs: 17 and 18; and    -   d) determining the sequences of the one or more amplicons; and    -   e) identifying the sample as:        -   i) containing or not containing the vaginal epithelial cell            based on the sequence of the amplicon corresponding to the            genetic locus corresponding to SEQ ID NO: 1 in the genomic            DNA isolated from the sample,        -   ii) containing or not containing the sperm based on the            sequence of the amplicon corresponding to the genetic locus            corresponding to SEQ ID NO: 6 in the genomic DNA isolated            from the sample,        -   iii) containing or not containing the blood cell based on            the sequence of the amplicon corresponding to the genetic            locus corresponding to SEQ ID NO: 11 in the genomic DNA            isolated from the sample, and/or        -   iv) containing or not containing the buccal epithelial cell            based on the sequence of the amplicon corresponding to the            genetic locus corresponding to SEQ ID NO: 16 in the genomic            DNA isolated from the sample.

In certain embodiments, the methods described herein to identify asample as containing a vaginal epithelial cell, sperm, blood cell, or abuccal epithelial cell are practiced on a forensic sample to detect thepresence of one or more of these cells in the forensic sample. Incertain embodiments, the methods are practiced on a forensic sample thatis processed to separate a cell suspected to be a vaginal epithelialcell, sperm, blood cell, or buccal epithelial cell before the step ofisolating the genomic DNA.

In one embodiment, the sequence of one or more amplicons correspondingto the genetic loci corresponding to SEQ ID NOs: 1, 6, 11, or 16 arecompared to a reference sequence to determine the methylation status ofthe genetic loci corresponding to SEQ ID NOs: 1, 6, 11, or 16, which inturn is used to identify the sample as containing or not containing oneor more cells selected from a vaginal epithelial cell, a sperm, a bloodcell, or a buccal epithelial cell.

A skilled artisan can design a sequencing primer to sequence theamplicons corresponding to the genetic loci corresponding to SEQ ID NOs:1, 6, 11, or 16 based on the sequences of these genetic loci and thesequences of the regions flanking these genetic loci in the genomic DNA.Such embodiments are within the purview of the invention.

In another embodiment, a sequencing primer can be designed based on anadapter introduced into the amplicon by incorporating the adapter intoone of the forward or reverse primers. An “adapter” as used herein is asequence of about 10 to 20 nucleotides that can be introduced into anamplicon by incorporating the adapter into the primer used for theamplification of the amplicon. Once an amplicon contains an adaptersequence, a primer designed based on the sequence of the adapter can beused to sequence the amplicon.

One embodiment of the invention provides a method for determining thelevels of methylation at genetic loci corresponding to one or more ofSEQ ID NOs: 1, 6, 11, and 16, in a genomic DNA from a cell, the methodcomprising the steps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, 11, and 16,        and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic loci corresponding to        one or more of SEQ ID NOs: 1, 6, 11, and 16.

In a specific embodiment, the invention provides a method fordetermining the levels of methylation at the genetic locus correspondingto SEQ ID NO: 1 in a genomic DNA from a cell, the method comprising thesteps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the genetic locus corresponding SEQ        ID NO: 1, and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic locus corresponding to        SEQ ID NO: 1.

Certain such embodiments further comprise determining the levels ofmethylation at genetic loci corresponding to one or more of SEQ ID NOs:6, 11, and 16, in a genomic DNA from a cell, the method comprising thesteps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 6, 11, and 16, and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic loci corresponding to        one or more of SEQ ID NOs: 6, 11, and 16.

In another embodiment, the invention provides a method for determiningthe levels of methylation at the genetic locus corresponding to SEQ IDNO: 6 in a genomic DNA from a cell, the method comprising the steps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the genetic locus corresponding SEQ        ID NO: 6, and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic locus corresponding to        SEQ ID NO: 6.

Certain such embodiments further comprise determining the levels ofmethylation at genetic loci corresponding to one or more of SEQ ID NOs:1, 11, and 16, in a genomic DNA from a cell, the method comprising thesteps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 11, and 16, and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic loci corresponding to        one or more of SEQ ID NOs: 1, 11, and 16.

In a further embodiment, the invention provides a method for determiningthe levels of methylation at the genetic locus corresponding to SEQ IDNO: 11 in a genomic DNA from a cell, the method comprising the steps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the genetic locus corresponding SEQ        ID NO: 11, and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic locus corresponding to        SEQ ID NO: 11.

Certain such embodiments further comprise determining the levels ofmethylation at genetic loci corresponding to one or more of SEQ ID NOs:1, 6, and 16, in a genomic DNA from a cell, the method comprising thesteps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, and 16, and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic loci corresponding to        one or more of SEQ ID NOs: 1, 6, and 16.

In an even further embodiment, the invention provides a method fordetermining the levels of methylation at the genetic locus correspondingto SEQ ID NO: 16 in a genomic DNA from a cell, the method comprising thesteps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the genetic locus corresponding SEQ        ID NO: 16, and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic locus corresponding to        SEQ ID NO: 16.

Certain such embodiments further comprise determining the levels ofmethylation at genetic loci corresponding to one or more of SEQ ID NOs:1, 6, and 11, in a genomic DNA from a cell, the method comprising thesteps of:

-   -   (a) isolating the genomic DNA from the cell,    -   (b) treating the genomic DNA with bisulfite,    -   (c) conducting a PCR using the bisulfite treated genomic DNA as        a template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 6, and 11, and    -   (d) analyzing the PCR amplicons produced in step c) to determine        the level of methylation at the genetic loci corresponding to        one or more of SEQ ID NOs: 1, 6, and 11.

The details described above regarding the techniques for determining thelevel of methylation at the genetic loci corresponding to one or moreSEQ ID NOs: 1, 6, 11, or 16 in the genomic DNA in a sample based on theanalysis of the amplicons produced in the PCR amplification steps arealso applicable to the method for determining the levels of methylationat these genetic loci. The primer pairs designed to amplify thesegenetic loci, sequencing primers and other details described above arealso applicable to the method for determining the levels of methylationat these genetic loci.

A further embodiment of the invention provides a kit comprising one ormore primer pairs designed to amplify the genetic loci corresponding toone or more SEQ ID NOs: 1, 6, 11, or 16 in a bisulfite treated humangenomic DNA. In one embodiment, the kit comprises one or more primerpairs selected from:

i) a primer pair having the sequences of SEQ ID NOs: 2 and 3,

ii) a primer pair having the sequences of SEQ ID NOs: 6 and 7,

iii) a primer pair having the sequences of SEQ ID NOs: 12 and 13, and

iv) a primer pair having the sequences of SEQ ID NOs: 17 and 18.

A skilled artisan can design additional primer pairs to amplify thegenetic loci corresponding to one or more SEQ ID NOs: 1, 6, 11, or 16based on the sequences of these genetic loci and the sequences offlanking regions in the genomic DNA and such embodiments are within thepurview of the invention.

In one embodiment, the invention provides a kit comprising a primer pairof SEQ ID NOs: 2 and 3, optionally, further comprising, one or moreprimer pairs selected from:

i) a primer pair having the sequences of SEQ ID NOs: 6 and 7,

ii) a primer pair having the sequences of SEQ ID NOs: 12 and 13, and

iii) a primer pair having the sequences of SEQ ID NOs: 17 and 18.

In another embodiment, the invention provides a kit comprising a primerpair of SEQ ID NOs: 6 and 7, optionally, further comprising, one or moreprimer pairs selected from:

i) a primer pair having the sequences of SEQ ID NOs: 2 and 3,

ii) a primer pair having the sequences of SEQ ID NOs: 12 and 13, and

iii) a primer pair having the sequences of SEQ ID NOs: 17 and 18.

In a further embodiment, the invention provides a kit comprising aprimer pair of SEQ ID NOs: 12 and 13, optionally, further comprising,one or more primer pairs selected from:

i) a primer pair having the sequences of SEQ ID NOs: 2 and 3,

ii) a primer pair having the sequences of SEQ ID NOs: 6 and 7, and

iii) a primer pair having the sequences of SEQ ID NOs: 17 and 18.

In an even further embodiment, the invention provides a kit comprising aprimer pair of SEQ ID NOs: 17 and 18, optionally, further comprising,one or more primer pairs selected from:

i) a primer pair having the sequences of SEQ ID NOs: 2 and 6,

ii) a primer pair having the sequences of SEQ ID NOs: 6 and 7, and

iii) a primer pair having the sequences of SEQ ID NOs: 12 and 13.

In further embodiments, the kit comprises one or more reagents, forexample, reagents for treating a sample, reagents for isolating cellsfrom the sample, reagents for isolating genomic DNA from the sample,reagents for bisulfite treating the genomic DNA, reagents for conductingPCR, and reagents for conducting pyrosequencing.

As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Further, to the extent that the terms “including,”“includes,” “having,” “has,” “with,” or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”The transitional terms/phrases (and any grammatical variations thereof)“comprising,” “comprises,” “comprise,” include the phrases “consistingessentially of,” “consists essentially of,” “consisting,” and“consists.”

The phrases “consisting essentially of” or “consists essentially of”indicate that the claim encompasses embodiments containing the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristic(s) of the claim.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within 1 or more than 1 standard deviation, per the practice inthe art. Alternatively, “about” can mean a range of up to 0-20%, 0 to10%, 0 to 5%, or up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed. In the context of compositions containingamounts of ingredients where the term “about” is used, thesecompositions contain the stated amount of the ingredient with avariation (error range) of 0-10% around the value (X±10%). In thecontext of melting temperatures where the term “about” is used, themelting temperatures are within 0.45° C. of the stated meltingtemperature.

As used herein, the term “level of methylation” or “methylation status”as applied to a genetic locus refers to whether one or more cytosineresidues present in a CpG have or do not have a methylation group. Thelevel of methylation or methylation status refers to the percentage ofcells in a sample that do or do not have a methylation group on suchcytosines. For example, if 50 cells in a pool of 100 cells containmethylated cytosines at a CpG site, the level of methylation ormethylation status of the CpG site is 50%.

A primer pair is a pair of oligonucleotides, each having about 15 to 25nucleotides, and designed to amplify a specific locus from template DNA.Guidelines for designing a primer pair to amplify a specific locus to ina template DNA are well known in the art.

A singleplex PCR is a reaction where only one set of primers is used perreaction; whereas, a multiplex reaction is one that uses multiple primersets per PCR reaction.

MATERIALS AND METHODS

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Designing Primers for Genomic Loci Specific for a Body Fluid

DNA from known body fluids are used in single reactions, i.e., in asingleplex. Initially primer pairs in singleplex PCR reactions aretested. The primers are designed herein to amplify a specific locus inthe genomic DNA regardless of methylation status of the genomic locus.Because amplification is independent of methylation, for body fluididentification the primers are designed to amplify a region thatpresents a different methylation status for the target body fluid whencompared to the other body fluids, allowing distinction by TM analysis(FIG. 1).

Multiplex analysis is performed with two, three, or four primer pairs.The primer pairs can be selected from:

i) a primer pair (VE_8) designed to amplify a specific locus in thegenomic DNA from vaginal epithelial cells,

ii) a primer pair (INPD200_TM60) designed to amplify a specific locus inthe genomic DNA from blood,

iii) a primer pair (ZC3H12D) designed to amplify a specific locus in thegenomic DNA from vaginal semen,

iv) a primer pair (VE_15) designed to amplify a specific locus in thegenomic DNA from saliva.

Multiplex reactions using two, three, or four primer pairs are used toobtain a melt curve with all the expected TMs (FIGS. 2-4).

Also, Table 1, below shows averages of melting temperature (T_(M), ° C.)and standard deviation (SD) for 10 samples of each body fluid and 4samples (INPP5D200_TM60) or 5 samples (VE_8) of each control amplifiedwith primers specific for the two loci INPP5D200_TM60 and VE_8. UnMmeans the unmethylated control and M represents the methylated control.

TABLE 1 Melting averages for cell specific loci disclosed herein. TargetBody T_(M)(° C.) ± SD Fluid Marker Name ‘cg’ code Blood Saliva Semen VEUnM M Blood INPP5D200_TM60 cg00438740 80.0 ± 0.13 83.0 ± 0.24 83.0 ±0.30 83.0 ± 0.45 80.0 ± 0.20 83.0 ± 0.17 VE VE_8 cg08751438 78.1 ± 0.1278.1 ± 0.11 78.1 ± 0.10 76.6 ± 0.16 76.5 ± 0.11 78.1 ± 0.06

REFERENCES

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We claim:
 1. A method for identifying a sample as containing or notcontaining a blood cell, the method comprising the steps of: (a)determining the level of methylation at the genetic locus of SEQ ID NO:11 in: i) a genomic DNA isolated from the sample, and ii) optionally, acontrol genomic DNA; (b) optionally, obtaining one or more referencevalues corresponding to the level of methylation at the genetic locus ofSEQ ID NO: 11; and (c) identifying the sample as containing, or notcontaining, the blood cell based on the level of methylation at thegenetic locus of SEQ ID NO: 11 in the genomic DNA isolated from thesample, the level of methylation at the genetic locus of SEQ ID NO: 11in the genomic DNA obtained from the sample being determined byhigh-resolution melt (HRM) analysis, HRM comprising the steps of: i)isolating the genomic DNA from the sample and optionally, the controlsample; ii) treating the isolated genomic DNA with bisulfite; iii)polymerase chain reaction (PCR) amplifying the genetic locus of SEQ IDNO: 11 to produce the corresponding amplicon, the PCR amplifying beingperformed using a primer pair comprising SEQ ID NOs: 12 and 13; and iv)determining the melting temperature of the amplicon; and v) identifyingthe sample as containing, or not containing, the blood cell based on themelting temperature of the amplicon corresponding to the genetic locusof SEQ ID NO: 11 in the genomic DNA isolated from the sample.
 2. Themethod of claim 1, wherein the control sample is obtained from a knownblood cell or a cell other than the blood cell known to have methylationlevel at SEQ ID NO: 11 to be different from the methylation level at SEQID NO: 11 in the known blood cell.
 3. The method of claim 1, wherein thecell other than the blood cell is a vaginal epithelial cell, a buccalcell, or a sperm.
 4. The method of claim 1, wherein the sample is aforensic sample.
 5. The method of claim 1, wherein the sample isprocessed to separate a cell suspected to be the blood cell before step(i) of isolating the genetic material.
 6. A method for determining thelevel of methylation at the genetic locus of SEQ ID NO: 11 in a genomicDNA isolated from a cell, the method comprising the steps of: i)isolating the genomic DNA from the cell; ii) treating the isolatedgenomic DNA with bisulfite; iii) PCR amplifying the genetic locus of SEQID NO: 11 to produce the corresponding amplicon, the PCR amplifyingbeing performed using a primer pair comprising SEQ ID NOs: 12 and 13;and iv) determining the melting temperature of the amplicon; and v)identifying the level of methylation at the genetic locus of SEQ ID NO:11 in the genomic DNA isolated from the cell based on the meltingtemperature of the amplicon produced in step iii).
 7. The method ofclaim 6, wherein the cell is isolated from a forensic sample.
 8. Themethod of claim 7, wherein the cell isolated from the forensic sample issuspected to be a blood cell.
 9. A kit comprising a primer comprisingSEQ ID NO: 12 and a primer comprising SEQ ID NO:
 13. 10. The kit ofclaim 9, further comprising one or mere primer pairs selected from: i)SEQ ID NOs: 2 and 3, ii) SEQ ID NOs: 7 and 8, and iii) SEQ ID NOs: 17and 18.